Bibliographic details of the publications referred to by author in this specification are collected alphabetically at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
Thalassemia is an inherited autosomal recessive blood disorder caused by the faulty synthesis of hemoglobin. This arises by one or more genetic defects affecting synthesis of the α- or β-globin chains which make up hemoglobin.
The World Health Organization (WHO) has conservatively estimated that approximately 7 percent of the world's population are carriers of various types of hemoglobinopathies, with an estimated 300,000 severely affected patients born worldwide each year. Although thalassemia is most common in Mediterranean, Middle Eastern, African and Asian populations (Olivieri (1999) N Engl J Med 341:99-109; Thein (2005) Hematology: 31-37), the ever rising rates of population migration mean this condition is encountered with increasing frequency in many parts of the world, including Northern Europe, North America and Australia. Thalassemia is fatal if left untreated and patients are dependent on a regular blood transfusion every 3-4 weeks for the rest of their lives.
There are over 200 β-globin gene mutations (Olivieri (1999) supra) which impair β-globin synthesis, resulting in imbalanced globin chain synthesis and β-thalassemia. In these situations, the excess, unbound α-globin chains precipitate in erythroid progenitor cells resulting in premature cell death, ineffective erythropoiesis and severe anemia. The key role of globin imbalance in contributing to thalassemia severity is most clearly illustrated in individuals who inherit an abnormal number of functional α-globin genes along with β-globin mutations. Individuals who co-inherit α-thalassemia with homozygous β-thalassemia have an improved phenotype and suffer less severe anemia than if either set of mutations was inherited alone (Camaschella et al. (1995) Am J Hematol 48:82-87; Cao et al (1991) Am J Pediatr Hematol Oncol. 13:179-188; Kanavakis et al. (2004) Blood Cells Mol Dis 32:319-324; Schrier (2002) Curr Opin Hematol 9:123-126; Thein (2005) Haematologica 90:649-660). The degree of correction is closely related to the degree to which globin chain balance has been restored (Thein et al (1984) Br J Haematol. 56:333-337). One mutated copy of α-globin generally has minimal impact but two or three mutated α-globin genes can improve β-thalassemic phenotypes significantly (Camaschella et al. (1995) supra; Cao et al. (1991) supra; Kanavakis et al. (2004) supra; Schrier (2002) supra; Thein (2005) supra). Therefore, alterations in α-globin chain synthesis can have considerable effects on β-thalassemic phenotypes and can even confer transfusion independence. Given that excess production of α-globin leads to widespread detrimental effects in β-thalassemia, reduction of α-globin synthesis would likely improve the β-thalassemic phenotype, raising the possibility of reducing α-globin expression as a therapy for β-thalassemia. The difficulty, however, is to specifically target α-globin gene expression.
Expression and synthesis of the α-globin and β-globin chains of hemoglobin is balanced during normal erythropoiesis and any disruptions in the α:β-globin synthesis ratios results in thalassemia (Olivieri (1999) supra; Thein (2005) supra). β-Thalassemia arises when α-globin is synthesized at levels exceeding the binding capacity of available β-globin chains, usually due to mutations affecting the β-globin locus which reduce β-globin expression (Schrier (1994) Annu Rev Med 45:211-218). Conversely, α-thalassemia occurs due to mutations which result in decreased α-globin expression, leading to an excess of β-globin chains (Schrier (1994) supra). Reduced expression of either of the globin chains leads to decreased formation of functional hemoglobin tetramers, yet this plays a relatively minor role in contributing to the severely anemic phenotype characteristic of the thalassemias. Instead, it is the damage caused at the cellular level by excess, improperly paired globin chains, which leads to premature cell death and accounts for the majority of the pathology (Schrier (1994) supra). Excess α-globin results in the formulation of large, insoluble aggregates which can be visualized by light microscopy in an estimated one third of β-thalassemic red blood cells (RBCs) [Fessas (1963) Blood 21:21-32], and occurs even in the earliest erythroid precursor cells (Schrier (1994) supra). These α-globin aggregates cause mechanical damage to membrane structures and trigger premature apoptosis in erythroid progenitor cells, leading to ineffective erythropoiesis (Thein (2005) supra; Kanavakis et al. (2004) supra). Furthermore, the excess α-globin is heavily oxidized (Advani et al. (1992) Blood 79:1064-1067) and each globin chain also carries a heme bound iron which can induce generation of reactive oxygen species (ROS) [Schrier et al. (2003) Redox Rep. 8:241-245]. It is believed that the increased ROS oxidizes adjacent membrane proteins, leading to severe membrane abnormalities and unstable cell membranes and this results ultimately in hemolysis and ungreatly exacerbating the anemic phenotype in β-thalassemia (Advani et al. (1992) supra).
RNAi is a highly conserved, naturally occurring mechanism of gene suppression found in plant, yeast and mammalian cells, which can be mediated by naturally occurring or synthetic short interfering RNAs (siRNA) [Hannon (2002) Nature 418:244-251]. In mammalian cells, RNAi can be induced using dsRNA of 19-21 bp with characteristic two nucleotide 3′ overhangs and 5′ phosphate groups. These are incorporated into an RNA induced silencing complex (RISC) and used as a template for cleavage of endogenous mRNA. Since targeting is dependent mainly on Watson-Crick base-pairing, it is theoretically possible to utilize this pathway to reduce expression of any gene in a sequence-specific manner.
Sarakul et al. (2008) Biochemical and Biosphysical Research Communication 369:935-938 disclosed the use of siRNA at a specific region of Exon2 of the α-globin gene locus. However, other target sites within the gene were ineffective in reducing expression. Chinese Patent Application No. 100567490 (CN 100567490) also used siRNA to target specific sites with variable effectiveness. These sites were defined as α2 and α3 (referred to herein as “CNα2” and “CNα3”, respectively). CNα3 targets codons 127 to 133 which is SEQ ID NO:3 in Sarakul et al. (2008) supra. The latter authors stated that only one sequence (SEQ ID NO:1, targeting codons 41 to 47 in Exon2) had any effect. Hence, there is clearly inconsistency between the data by Sarakul et al. (2008) supra and CN100567490. This highlights the difficulty in designing effective siRNA molecules. The present disclosure included siRNA encoding SEQ ID NO:1, (targeting codons 41 to 47 in Exon2) as a control (referred to as Hs-siα5) and found that this siRNA was the least effective in reducing alpha-globin expression compared to all other siRNA tested. Also demonstrating that siRNA targeting other regions not anticipated by Sarakul et al. (2008) supra and CN100567490 are potentially more effective in reducing α-globin.
In order to assess the feasibility of RNAi-mediated therapy, a mouse model has been developed. The most well characterized is the heterozygous β-globin knockout (β-KO) model (Yang et al. (1995) Proc Natl Acad Sci USA 92:11608-11612), which displays distinct hematological abnormalities consistent with β-thalassemia (wide variations of red cell distribution width (RDWs), significant reductions in hemoglobin (Hb) and hematocrit (HCT) levels) [Yang et al. (1995) supra; Beauchemin et al. (2004) J Biol chem. 279:19471-19480; Vadolas et al. (2005) Biochim Biophys Acta 1728:150-162). In order to assess the effects of reduced α-globin expression in β-thalassemic mice, heterozygous α-globin knockout mice (α-KO) were crossed with thalassemic β-KO mice (Beauchemin et al. (2004) supra; Al-Hasani et al. (2004) Transgenic Res 13:235-243; Paszty et al. (1995) Nat Genet 11:33-39). The resultant double heterozygous (DH) α-KO/β-KO progeny expressed reduced, but balanced, levels of both α-globin and β-globin and displayed a normal range of RDWs with Hb and HCT almost completely restored to wild type (WT) levels. Furthermore, the reduced drive for extramedullary erythropoiesis combined with reduced clearance of damaged RBC, resulted in a marked reduction in spleen size indistinguishable from those found in WT mice.
Whilst intuitively there are benefits of reducing α-globin expression in the context of β-thalassemia, there have been no reported substantial reductions of α-globin using methods which are conducive to therapy in humans. An siRNA approach was investigated to mediate reductions in α-globin expression in mice. One highly effective siRNA sequence (siα4), located in the 3′ untranslated region, was demonstrated to reduce α-globin expression in hemoglobinized murine erythroleukemic (MEL) cells by approximately 65% at both the RNA and the protein levels (Voon et al. (2008) Haematologica 93:1238-1242). The efficacy of siα4 was further confirmed by testing this sequence in primary cultures of erythropoietic progenitor cells
There is a need to use gene silencing technology to greater effect to treat β-thalassemia and related conditions arising from an excess amount of α-globin in humans.